Clinical Bladder Cancer Specimens Research Articles

Y-Box Binding Protein 1 Regulates Angiogenesis in Bladder Cancer via miR-29b-3p-VEGFA Pathway.

Angiogenesis plays a vital role in the development of bladder cancer (BC). The Y-box-binding protein 1 (YB-1) is a well-known oncoprotein which is closely related to angiogenesis of tumors, but the relationship and mechanism of YB-1 and angiogenesis in BC remain unclear. Based on 56 clinical BC specimens, this study found that high expression of YB-1 samples demonstrated a higher expression of vascular endothelial growth factor A (VEGFA) than those of YB-1 low expression. Subsequently, the expression of YB-1 and miR-29b-3p was regulated in the BC cell lines where we noted that YB-1 promoted VEGFA expression by downregulating the expression of miR- 29b-3p. The ability of BC cells to induce angiogenesis decreased after YB-1 was knocked down. Moreover, the in vivo study further confirmed that YB-1 promotes angiogenesis in BC. Our findings enhance the understanding of how YB-1 promotes angiogenesis in BC and provide evidence for YB-1 as a therapeutic target of BC. Moreover, this may provide new inspiration for miRNAs replacement therapies.

MiR-21 Overexpression Promotes Esophageal Squamous Cell Carcinoma Invasion and Migration by Repressing Tropomyosin 1

The migration and invasion of esophageal squamous cell carcinoma are associated with clinical outcomes, however, the mechanisms remain poorly understood. Here, we found that miR-21 is significantly overexpressed in ESCC, lung cancer, and bladder cancer compared with the adjacent normal tissue. MiR-21 and TPM1 expressions were analyzed by RT-qPCR and WB in 30 ESCC, 10 lung cancer, and 10 bladder cancer clinical specimens, each with matched adjacent normal tissue. Knockdown and overexpression of miR-21 as well as knockdown of TPM1 in ESCC cell lines were performed using synthetic oligonucleotides. TPM1 3′UTR luciferase reporter constructs were used to investigate targeting of TPM1 by miR-21. ESCC migration and invasion were assessed using transwell migration and invasion assays. Inhibition of miR-21 reduced migration and invasion in two ESCC cell lines, and overexpression of miR-21 promoted migration and invasion in vitro. Interestingly, TPM1 exhibited inverse patterns of expression compared with miR-21 in tissues and cell lines. Luciferase reporter assays demonstrated that TPM1 was directly regulated by miR-21. Moreover, the forced overexpression of miR-21 repressed the TPM1 expression, while silencing of miR-21 restored the TPM1 expression in ESCC cell lines. What is more, simultaneous silencing of miR-21 and TPM1 expressions did not alter the migratory and invasive characteristics demonstrating that the effects of miR-21 were mediated through TPM1. In conclusion, the aberrant overexpression of miR-21 is common in cancer and promotes the migration and invasion of ESCC through inhibiting the TPM1 expression. These results suggest that miR-21 may be a novel predictive marker and therapeutic target for treatment of ESCC.

MiR-425 Promotes Migration and Invasion in Bladder Cancer by Targeting Dickkopf 3.

Background: Bladder cancer (BC) is a common malignancy with high morbidity and mortality. MicroRNAs (miRNAs) are critical post-transcriptional regulators in various cancers. This study aimed to investigate the effect of miR-425 on the migration and invasion of BC.Methods: The expression of miR-425 and Dickkopf 3 (DKK3) was examined in clinical BC specimens. T24 and 5637 BC cell lines were employed and transfected with miR-425 inhibitors. The correlation between miR-425 and DKK3 was determined by a luciferase reporter assay. Cell migration and invasion capacity were measured by wound healing and Transwell assays. The expression levels of DKK3, E-cadherin, N-cadherin and vimentin were analysed by Western blotting and qRT-PCR.Results: miR-425 was negatively correlated with the expression of DKK3 in clinical BC specimens. Further studies identified DKK-3 as a direct target of miR-425. Moreover, knockdown of miR-425 promoted the expression of DKK3 and suppressed cell migration and invasion capacity. miR-425 silencing increased E-cadherin levels but decreased vimentin and N-cadherin protein levels in T24 and 5637 cells.Conclusion: Our study indicated that miR-425 promoted the migration and invasion of BC via targeting DKK3.

Predictive value of cadherin-11 for subsequent recurrence and progression in non-muscle invasive bladder cancer.

Cadherin-11 (CDH11) is a type II cadherin and reported to function as an oncogene in various cancers. Our present study aims to investigate the role of CDH11 in bladder cancer (BCA). Bioinformatics analysis was performed in four independent microarray data including 56 non-muscle-invasive bladder cancer (NMIBC) and 132 muscle-invasive bladder cancer (MIBC) tissues from Gene Expression Omnibus to screen out differentially expressed genes. Next, we detected CDH11 expression in BCA specimens and cell lines by qPCR and western blotting assays. Immunohistochemical analyses were performed in 209 paraffin-embedded BCA samples and 30 adjacent normal bladder tissues. Bioinformatics analysis revealed that CDH11 had a higher expression level in MIBC tissues than in NMIBC, which was consistent with our clinical BCA specimens and cell lines at both mRNA and protein levels. Immunohistochemical analysis demonstrated that over-expression of CDH11 was closely related to the histological grade, pT status, tumour size and poor outcomes of BCA patients. What's more, CDH11 (area under curve (AUC)=0.673 and 0.735) had a better predictive value than E-cadherin (AUC=0.629 and 0.629) and a similar discrimination with the European Organization for Research and Treatment of Cancer (EORTC) score system (AUC=0.719 and 0.667) in evaluating potential recurrence and progression of NMIBC. Moreover, combination of CDH11 and EORTC score system was the best predictive model in predicting recurrence of NMIBC (AUC=0.779) among the three models. CDH11 was a reliable therapeutic target in BCA and a useful index to predict the possibilities of recurrence and progression in NMIBC patients.

CircPTPRA acts as a tumor suppressor in bladder cancer by sponging miR-636 and upregulating KLF9.

Growing evidence suggests that circular RNAs (circRNAs) play pivotal roles in cancer progression. In this study, bioinformatic analysis identified a dysregulated circRNA termed circPTPRA in bladder cancer (BC). By using qRT-PCR analysis, we verified that circPTPRA is down-regulated in clinical BC specimens compared with the matched non-tumor samples, while correlation analyses showed that low circPTPRA expression is associated with poor prognosis, advanced tumor stage and larger tumor size. Based on these findings, we conducted functional assays and revealed that circPTPRA inhibits BC cell proliferation in vitro and tumor growth in vivo. In addition, RNA pull-down, miRNA capture, FISH, and luciferase reporter assays demonstrated that circPTPRA can directly sponge miR-636. Cell transfection experiments showed that miR-636 promotes the proliferation of BC cells by decreasing the expression of Krüppel Like Factor 9 (KLF9) upon binding to the 3’UTR of its mRNA.Further analysis confirmed that circPTPRA competitively sponges miR-636 to upregulate the KLF9 expression, leading to decreased proliferation of BC cells. Our investigation indicates that circPTPRA acts as a tumor suppressor in BC, and suggests that this circRNA may be a novel prognostic biomarker and therapeutic target in BC.

Aberrantly expressed PLOD1 promotes cancer aggressiveness in bladder cancer: a potential prognostic marker and therapeutic target.

Bladder cancer (BC) is the ninth most malignant tumor worldwide. Some BC patients will develop muscle‐invasive BC (MIBC), which has a 5‐year survival rate of approximately 60% due to metastasis. As such, there is an urgent need for novel therapeutic and diagnostic targets for MIBC. Analysis of novel antitumor microRNA (miRNA)‐mediated cancer networks is an effective strategy for exploring therapeutic targets and prognostic markers in cancers. Our previous miRNA analysis revealed that miR‐140‐5p acts as an antitumor miRNA in BC cells. Here, we investigated miR‐140‐5p regulation of BC molecular pathogenesis. Procollagen‐lysine, 2‐oxoglutarate 5‐dioxygenase 1 (PLOD1) was found to be directly regulated by miR‐140‐5p, and aberrant expression of PLOD1 was observed in BC clinical specimens. High PLOD1 expression was significantly associated with a poor prognosis (disease‐free survival: P = 0.0204; overall survival: P = 0.000174). Multivariate analysis showed PLOD1 expression to be an independent prognostic factor in BC patients (hazard ratio = 1.51, P = 0.0099). Furthermore, downregulation of PLOD1 by siRNAs and a specific inhibitor significantly decreased BC cell aggressiveness. Aberrant expression of PLOD1 was closely associated with BC pathogenesis. In summary, the present study showed that PLOD1 may be a potential prognostic marker and therapeutic target for BC.

Tumor‑suppressive microRNA‑223 targets WDR62 directly in bladder cancer.

miRNA‑223 (miR‑223) has been reported to function not only as a tumor suppressor, but also as an oncogenic microRNA (miRNA or miR) in various cancer cells. Therefore, the functional role of miR‑223 has not been elucidated to date, at least to the best of our knowledge. We previously performed the deep sequencing analysis of clinical bladder cancer(BC) specimens. It was revealed that miR‑223 expression was significantly downregulated in BC, suggesting that miR‑223 functions as a tumor suppressor miRNA in BC. The aim of this study was to investigate the functional roles of miR‑223 and to identify its targets in BC. The expression levels of miR‑223 were significantly decreased in our clinical BC specimens. The Cancer Genome Atlas (TCGA) database indicated that miR‑223 expression was related to lymphovascular invasion and distant metastasis. The restoration of miR‑223 expression significantly inhibited tumor aggressiveness and induced apoptosis via caspase‑3/7 activation in BC cells. WD repeat domain62(WDR62), a candidate target of miR‑223 according to insilico analyses, has been previously proposed to play a role in neurodevelopment. Direct binding between WDR62 and miR‑223 was confirmed by luciferase assay. The TCGA database revealed positive associations between WDR62 mRNA expression and a higher tumor grade and stage in BC. The knockdown of WDR62 significantly inhibited tumor aggressiveness and induced the apoptosis of BC cells. On the whole, the findings of this study reveal a novel miR‑223 target, oncogenic WDR62, and provided insight into the oncogenesis of BC.

Abstract 518: Dual strands of pre-miR-223 (miR-223-5p and miR-223-3p) possess antitumor roles and these are involved in bladder cancer pathogenesis

Abstract Bladder cancer (BC) is a common urologic cancer and the ninth most common cause of death worldwide. In 2015, it was estimated that approximately 76,000 new cases were diagnosed as BC and 1600 patients died in United States of America. Approximately 70-80% of patients are diagnosed with non-muscle-invasive BC (NMIBC) at the first visit and high recurrence rates (50%-70%) are observed in this patients. Moreover, 15% of recurrent BC progress to muscle-invasive BC (MIBC) and 5-year survival rate for patients with MIBC is only approximately 60%. At present, there are no effective treatments for patients with advanced BC or failure of first-line treatment. MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules (19-22 bases in length) that act as fine-tuners for RNA expression in a sequence-specific manner. Unique nature of miRNA is a single miRNAs might be controlled a large number of protein-coding or protein noncoding RNAs in cells. Abnormal expression of miRNAs will be caused disruption of intracellular RNA networks and these events are involved in human diseases. In this study, we focused on both strands of pre-miR-223 (miR-223-5p and miR-223-3p) based on miRNA signature of bladder cancer (BC) and to investigate the functional significance of these miRNA in BC cells. Ectopic expression of these miRNAs showed that as in miR-223-3p (guide strand), miR-223-5p (passenger strand) inhibited the abilities of cancer cell migration and invasion in BC cells. Combination of gene expression and in silico database analyses demonstrated that a total of 33 putative targets were identified as miR-223-5p regulation in BC cells. Among these target genes, high expression of 5 genes (ANLN, HMMR, CDC20, GJB2 and BUB1B) were significantly associated with poor prognosis of the patients with BC by The Cancer Genome Atlas (TCGA) database analyses. Moreover, knockdown of ANLN significantly inhibited cell proliferation, migration, and invasion (P < 0.0001). The ANLN was highly expressed in BC clinical specimens. These results indicated that ANLN acts as an oncogene in BC. Aberrant expressed ANLN was detected in BC clinical specimens and its high expression was deeply involved in BC pathogenesis. Involvement of passenger strands of miRNAs in cancer pathogenesis is a novel concept of cancer research and novel miRNAs-mediated molecular networks may lead to a better understanding of BC pathogenesis and the development of new treatment protocols. Citation Format: Sho Sugawara, Yasutaka Yamada, Takayuki Arai, Atsushi Okato, Mayuko Kato, Tomohiko Ichikawa, Naohiko Seki. Dual strands of pre-miR-223 (miR-223-5p and miR-223-3p) possess antitumor roles and these are involved in bladder cancer pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 518.

Dual strands of the miR-223 duplex (miR-223-5p and miR-223-3p) inhibit cancer cell aggressiveness: targeted genes are involved in bladder cancer pathogenesis.

Analyses of microRNA (miRNA) expression signatures obtained by RNA sequencing revealed that some passenger miRNAs (miR-144-5p, miR-145-3p, miR-149-3p, miR-150-3p, and miR-199a-3p) acted as anti-tumor miRNAs in several types of cancer cells. The involvement of passenger strands in the pathogenesis of human cancer is a novel concept. Based on the miRNA signature of bladder cancer (BC) obtained by RNA sequencing, we focused on both strands of the miR-223-duplex (miR-223-5p and miR-223-3p) and investigated their functional significance in BC cells. Ectopic expression of these miRNAs showed that both miR-223-3p (the guide strand) and miR-223-5p (the passenger strand) inhibited cancer cell migration and invasion of BC cells. The role of miR-223-5p (the passenger strand) has not been well studied. Combining gene expression studies and in silico database analyses, we demonstrated the presence of 20 putative target genes that could be regulated by miR-223-5p in BC cells. Among these targets, high expression of five genes (ANLN, INHBA, OIP5, CCNB1, and CDCA2) was significantly associated with poor prognosis of BC patients based on The Cancer Genome Atlas (TCGA) database. Moreover, we showed that a gene (ANLN) encoding a multifunctional actin-binding protein was directly regulated by miR-223-5p in BC cells. Overexpression of ANLN was observed in BC clinical specimens and high expression of ANLN was significantly associated with poor prognosis of BC patients. We suggest that studies of regulatory cancer networks, including the passenger strands of miRNAs, may provide new insights into the pathogenic mechanisms of BC.

AMPKα2 Regulates Bladder Cancer Growth through SKP2-Mediated Degradation of p27.

These results highlight the contribution of AMPKα2 as a mechanism for controlling bladder cancer growth by regulating proliferation through mTOR suppression and induction of p27 protein levels, thus indicating how AMPKα2 loss may contribute to tumorigenesis. Mol Cancer Res; 14(12); 1182-94. ©2016 AACR.

GON4L Drives Cancer Growth through a YY1-Androgen Receptor-CD24 Axis.

In principle, the inhibition of candidate gain-of-function genes defined through genomic analyses of large patient cohorts offers an attractive therapeutic strategy. In this study, we focused on changes in expression of CD24, a well-validated clinical biomarker of poor prognosis and a driver of tumor growth and metastasis, as a benchmark to assess functional relevance. Through this approach, we identified GON4L as a regulator of CD24 from screening a pooled shRNA library of 176 candidate gain-of-function genes. GON4L depletion reduced CD24 expression in human bladder cancer cells and blocked cell proliferation in vitro and tumor xenograft growth in vivo Mechanistically, GON4L interacted with transcription factor YY1, promoting its association with the androgen receptor to drive CD24 expression and cell growth. In clinical bladder cancer specimens, expression of GON4L, YY1, and CD24 was elevated compared with normal bladder urothelium. This pathway is biologically relevant in other cancer types as well, where CD24 and the androgen receptor are clinically prognostic, given that silencing of GON4L and YY1 suppressed CD24 expression and growth of human lung, prostate, and breast cancer cells. Overall, our results define GON4L as a novel driver of cancer growth, offering new biomarker and therapeutic opportunities. Cancer Res; 76(17); 5175-85. ©2016 AACR.

Abstract 1105: Dual tumor suppressors miR-139-5p/-3p derived from pre-miR-139 via targeting matrix metalloprotease 11 (MMP11) in bladder cancer

Abstract Introduction & Objective: The molecular mechanisms of recurrence and muscle invasion process of bladder cancer (BC) are not well understood. Therefore, understanding the molecular mechanisms of metastatic pathways underlying advanced BC using currently available genomic approaches might improve therapies for and prevention of the disease. Our recent study of microRNA (miRNA) expression signature of BC by deep-sequencing revealed that miR-139-5p (guide strand) and miR-139-3p (passenger strand) were significantly downregulated in BC tissues. In miRNA biogenesis, the passenger strand of miRNA duplex is discarded, while the guide strand is retained to direct recruitment of the RISC to target mRNAs. We hypothesis that these miRNAs act as tumor suppressors in BC. The aim of the present study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells. Material & Methods: Expression levels of miR-139-5p/-3p and its candidate target genes were validated in two BC cell lines (T24 and BOY) and BC clinical specimens (62 BCs and 23 normal bladder epitheliums: NBEs) by real-time PCR methods. Gain-of-function study, cell proliferation, migration, and invasion analyses were performed by using miR-139-5p/-3p transfected BC cell lines. In silico database and genome-wide gene expression analyses were applied for identification of miR-139-5p/-3p targets. Loss-of-function study for its target gene was performed by using siRNAs. Luciferase reporter analyses were employed to validate direct binding between the target gene and miR-139-5p/-3p. Overall patient survival between high and low expression of the target gene was analyzed by the Kaplan-Meier method. Results: The expression levels of miR-139-5p and miR-139-3p were significantly reduced in tumor tissues and BC cell lines compared with NBEs (P< 0.0001). Restoration of miR-139-5p or miR-139-3p in cancer cells revealed that both miRNAs inhibited cancer cell migration and invasion. Our data demonstrated that the gene coding for matrix metalloprotease 11 (MMP11) was a direct target of miR-139-5p/-3p regulation. Silencing of MMP11 inhibited cell migration and invasion in BC cells. Moreover, Kaplan-Meier analysis revealed that the patients with high MMP11 expression had lower overall survival probabilities than those with low expression (P = 0.043). Conclusions: Aberrant expressed novel miRNAs were identified by deep sequencing of BC miRNA signature. Two mature miRNAs, miR-139-5p and miR-139-3p derived from pre-miR-139 act as tumor-suppressors in BC cells. To the best of our knowledge, this is the first report demonstrating that dual tumor-suppressive miR-139-5p/-3p directly regulate MMP11 in BC cells. The identification of novel target gene regulated by the dual tumor-suppressors may lead to a better understanding of BC and the development of new therapeutic strategies to treat this disease. Citation Format: Masaya Yonemori, Naohiko Seki, Ryosuke Matsusihta, Kazutaka Miyamoto, Hirofumi Yoshino, Yusuke Goto, Mayuko Kato, Akira Kurozumi, Msayuki Nakagawa, Hideki Enokida. Dual tumor suppressors miR-139-5p/-3p derived from pre-miR-139 via targeting matrix metalloprotease 11 (MMP11) in bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1105.

Association of epithelial-mesenchymal transition and nuclear cofilin with advanced urothelial cancer

Tumor epithelial cells undergo a morphologic shift through the process of EMT with characteristic loss of cell polarity, conferring invasive and metastatic properties during cancer progression. Signaling by transforming growth factor-β mediates EMT programming and its phenotypic reversal to mesenchymal-epithelial transition. The role of EMT in bladder cancer progression to advanced disease is poorly understood. In this study, we conducted a retrospective analysis of the EMT landscape and actin cytoskeleton remodeling in a series of human bladder cancer specimens. Immunoreactivity for E-cadherin, N-cadherin, and vimentin protein expression was performed toward establishing an EMT signature in human bladder cancer. Serial sections were assessed for the primary regulator of the actin cytoskeleton remodeling and transforming growth factor-β signaling effector, cofilin. Our results demonstrate that EMT induction in clinical bladder cancer specimens is significantly associated with bladder cancer progression to high-grade, invasive disease. Evaluation of expression and cellular localization of the cytoskeleton regulator cofilin revealed a significant association between overexpression of nuclear cofilin with bladder cancer progression. This study is of translational significance in defining the value of EMT signature and cytoskeletal cofilin as potential tumor markers and targetable platforms for the treatment of invasive bladder cancer.

Regulation of UHRF1 by dual-strand tumor-suppressor microRNA-145 (miR-145-5p and miR-145-3p): Inhibition of bladder cancer cell aggressiveness.

In microRNA (miRNA) biogenesis, the guide-strand of miRNA integrates into the RNA induced silencing complex (RISC), whereas the passenger-strand is inactivated through degradation. Analysis of our miRNA expression signature of bladder cancer (BC) by deep-sequencing revealed that microRNA (miR)-145-5p (guide-strand) and miR-145-3p (passenger-strand) were significantly downregulated in BC tissues. It is well known that miR-145-5p functions as a tumor suppressor in several types of cancer. However, the impact of miR-145-3p on cancer cells is still ambiguous. The aim of the present study was to investigate the functional significance of miR-145-3p and BC oncogenic pathways and targets regulated by miR-145-5p/miR-145-3p. Ectopic expression of either miR-145-5p or miR-145-3p in BC cells significantly suppressed cancer cell growth, migration and invasion and it also induced apoptosis. The gene encoding ubiquitin-like with PHD and ring finger domains 1 (UHRF1) was a direct target of these miRNAs. Silencing of UHRF1 induced apoptosis and inhibited cancer cell proliferation, migration, and invasion in BC cells. In addition, overexpressed UHRF1 was confirmed in BC clinical specimens, and the high UHRF1 expression group showed a significantly poorer cause specific survival rate in comparison with the low expression group. Taken together, our present data demonstrated that both strands of miR-145 (miR-145-5p: guide-strand and miR-145-3p: passenger-strand) play pivotal roles in BC cells by regulating UHRF1. The identification of the molecular target of a tumor suppressive miRNAs provides novel insights into the potential mechanisms of BC oncogenesis and suggests novel therapeutic strategies.

MP88-01 DUAL TUMOR-SUPPRESSORS MIR-139-5P/-3P DERIVED FROM PRE-MIR-139 VIA TARGETING MATRIX METALLOPROTEASE 11 (MMP11) IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology V1 Apr 2016MP88-01 DUAL TUMOR-SUPPRESSORS MIR-139-5P/-3P DERIVED FROM PRE-MIR-139 VIA TARGETING MATRIX METALLOPROTEASE 11 (MMP11) IN BLADDER CANCER Masaya Yonemori, Naohiko Seki, Ryosuke Matsushita, Kazutaka Miyamoto, Hirofumi Yoshino, Yusuke Goto, Mayuko Kato, Akira Kurozumi, Masayuki Nakagawa, and Hideki Enokida Masaya YonemoriMasaya Yonemori More articles by this author , Naohiko SekiNaohiko Seki More articles by this author , Ryosuke MatsushitaRyosuke Matsushita More articles by this author , Kazutaka MiyamotoKazutaka Miyamoto More articles by this author , Hirofumi YoshinoHirofumi Yoshino More articles by this author , Yusuke GotoYusuke Goto More articles by this author , Mayuko KatoMayuko Kato More articles by this author , Akira KurozumiAkira Kurozumi More articles by this author , Masayuki NakagawaMasayuki Nakagawa More articles by this author , and Hideki EnokidaHideki Enokida More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2419AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The 5-year survival rate for patients with muscle invasive bladder cancer (BC) is approximately 60%, and patients with lymph node metastases die in the first 5 years after diagnosis. To improve the survival rate for patients with advanced BC, novel treatment strategies based on the current genomic approaches are needed. Our recent study of microRNA (miRNA) expression signature of BC by deep-sequencing revealed that miR-139-5p (guide strand) and miR-139-3p (passenger strand) derived from pre-miR-139 were significantly downregulated in BC tissues. In miRNA biogenesis, during RISC loading the passenger strand of miRNA duplex is discarded, while the guide strand is retained to direct recruitment of the RISC to target mRNAs. We hypothesis that these miRNAs function as tumor suppressors in BC. The aim of the present study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells. METHODS Expression levels of miR-139-5p/-3p and its candidate target genes were evaluated in two BC cell lines (T24 and BOY) and BC clinical specimens (62 BCs and 23 normal bladder epitheliums: NBEs) by qRT-PCR methods. For the gain of function study, proliferation, migration, and invasion were performed in miR-139-5p/-3p transfected BC cell lines. Putative target genes were determined by the analyses with GEO database and miR139-5p/-3p transfected T24 microarray study. Loss of function study for its target gene was examined. Overall survival between high and low expression of the target gene was analyzed by the Kaplan-Meier method. RESULTS The expression levels of miR-139-5p/-3p were reduced in tumor tissues and BC cell lines compared with NBEs (P < 0.0001). Restoration of miR-139-5p or miR-139-3p in cancer cells revealed that both miRNAs inhibited cancer cell migration and invasion. Our data demonstrated that MMP11 was a direct target. Silencing of MMP11 inhibited cell migration and invasion by BC cells. Moreover, Kaplan-Meier analysis revealed that the patients with high MMP11 expression had lower overall survival probabilities than those with low expression (p=0.043). CONCLUSIONS To the best of our knowledge, this is the first report demonstrating that dual tumor-suppressive roles of the miR-139-5p and miR-139-3p in BC cells, and these miRNAs directly regulated MMP11. The identification of target gene regulated by novel tumor-suppressive miRNAs may lead to a better understanding of molecular mechanisms of BC metastatic pathways and the development of new therapeutic strategies to treat this disease. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1129 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Masaya Yonemori More articles by this author Naohiko Seki More articles by this author Ryosuke Matsushita More articles by this author Kazutaka Miyamoto More articles by this author Hirofumi Yoshino More articles by this author Yusuke Goto More articles by this author Mayuko Kato More articles by this author Akira Kurozumi More articles by this author Masayuki Nakagawa More articles by this author Hideki Enokida More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract A20: Innovative drug discovery for bladder cancer by miR-130 family seed-targeting locked nucleic acid

Abstract Bladder cancer causes an estimated 150,000 deaths per year worldwide. Although 15% of the recurrent bladder cancer becomes an invasive type, targeted therapy for malignant bladder cancer does not make much progress. To investigate a novel therapeutic target, we focused on miRNA expression signatures obtained from bladder cancer clinical specimens, and found that the miR-130 family (miR-130b, miR-301a and miR-301b) was significantly up-regulated in bladder cancer specimens compared to adjacent normal urothelial specimens. Functional analyses revealed that the miR-130 family promoted bladder cancer cell migration and invasion by regulating both focal adhesion kinase (FAK) and Akt phosphorylation. Moreover, miR-130b directly targeted Phosphatase and Tensin Homolog Deleted from Chromosome 10 (PTEN), which suppresses FAK and Akt activation. Furthermore, Argonaute2-immunoprecipitation and the subsequent gene array analysis identified tyrosine-protein phosphatase non-receptor type 11 (PTPN11) as another target gene of both miR-130b and miR-301b. Thus, the miR-130 family promotes bladder cancer cell motility by regulating these target genes. We next designed miR-130 family-targeted LNA (Locked Nucleic Acid) that can inhibit entire miR-130 family through a common seed sequence. miR-130 family-targeted LNA suppressed bladder cancer cell proliferation, migration and invasion powerfully by targeting both PTEN and PTPN11. Furthermore, miR-130 family-targeted LNA significantly inhibits bladder tumor growth in a murine xenograft model. Taken together, the miR-130 family has a crucial role for malignant progression of bladder cancer and thus miR-130 family-targeted LNA could be a promising molecular-targeted drug for bladder cancer. Citation Format: Hiroshi Egawa, Kentaro Jingushi, Yuko Ueda, Kaori Kitae, Wataru Nakata, Kazutoshi Fujita, Motohide Uemura, Norio Nonomura, Kazutake Tsujikawa. Innovative drug discovery for bladder cancer by miR-130 family seed-targeting locked nucleic acid. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A20.

Abstract B15: MicroRNA-145/145* as a dual tumor-suppressor targeting UHRF1 in bladder cancer

Abstract Background: Bladder cancer (BC) can be classified into two categories: non-muscle-invasive BC (NMIBC) and muscle-invasive BC (MIBC). Although 70%–80% of patients are diagnosed with NMIBC, high recurrence rates (50%–70%) are observed in these patients. Moreover, among recurrent cases, 15% of BCs progress to MIBC disease. The five year survival rate for patients with NMIBC is close to 90%, whereas that for patients with MIBC is only approximately 60%. Patients with advanced BC are generally treated with combination chemotherapy consisting of gemcitabine and cisplatin, but progression-free survival is of limited duration. There are currently no effective second-line chemotherapeutic agents for advanced BC. The molecular mechanisms of recurrence and muscle invasion process of BC are not well understood. Therefore, understanding the molecular mechanisms of metastatic pathways underlying advanced BC using currently available genomic approaches might improve therapies for and prevention of the disease. Our recent study of microRNA (miRNA) expression signature of BC by deep-sequencing revealed that microRNA-145 (miR-145) and microRNA-145* (miR-145*) were significantly down regulated in BC tissues. In miRNA biogenesis, during RISC loading one strand (passenger strand) of miRNA duplex is discarded, while the other strand (guide strand) is retained to direct recruitment of the RISC to target mRNAs. In generally, miR-145* recognized as a passenger strand of pre-miR-145. We hypothesis that these miRNAs function as tumor suppressors in BC. The aim of the present study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells. Methods: Expression levels of miR-145, miR-145* and its candidate target gene were evaluated in BC cell lines (T24 and BOY) and BC clinical specimens (71 BCs and 12 normal bladder epitheliums: NBEs) by qRT-PCR methods. The functional studies of miR-145, miR-145* and target gene were performed to investigate cell proliferation, migration and invasion by cancer cell lines. To identify miR-145/145*-regulated molecular targets, genome-wide gene expression analysis, in silico database analysis, and dual-luciferase reporter assays were applied. Results: The expression levels of miR-145 and miR-145* were significantly reduced in tumor tissues and BC cell lines compared with NBEs (P &amp;lt; 0.0001). The expression levels of miR-145 and miR-145* were analysed for their correlation with one another.Restoration of miR-145 or miR-145* in cancer cells revealed that both miRNAs significantly inhibited cancer cell proliferation, migration and invasion. Our data demonstrated that the gene coding for Ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1) was a direct target of miR-145/145* regulation. Moreover, silencing of UHRF1 significantly inhibited cell proliferation, migration and invasion by bladder cancer cells. Conclusion: Down-regulation of the miR-145 and miR-145* were frequently observed in BC cells, and these miRNAs significantly inhibited cancer cell proliferation, migration and invasion, suggesting act as tumor-suppressors in BC. To the best of our knowledge, this is the first report demonstrating that dual tumor-suppressive miR-145/145* directly regulated UHRF1 in BC cells. The identification of novel target gene regulated by dual tumor-suppressor miR-145/145* may lead to a better understanding of BC and the development of new therapeutic strategies to treat this disease. Citation Format: Ryosuke Matsushita, Naohiko Seki, Hirofumi Yoshino, Yusuke Goto, Kazutaka Miyamoto, Masaya Yonemori, Hideki Enokida, Masayuki Nakagawa. MicroRNA-145/145* as a dual tumor-suppressor targeting UHRF1 in bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B15.

The miR-130 family promotes cell migration and invasion in bladder cancer through FAK and Akt phosphorylation by regulating PTEN.

Bladder cancer causes an estimated 150,000 deaths per year worldwide. Although 15% of the recurrent bladder cancer becomes an invasive type, currently used targeted therapy for malignant bladder cancer is still not efficient. We focused on the miR-130 family (miR-130b, miR-301a, and miR-301b) that was significantly upregulated in bladder cancer specimens than that of the normal urothelial specimens. We analyzed the functional significance of miR-130 family using a 5637 bladder cancer cell line and revealed that miR-130 family of inhibitors suppressed cell migration and invasion by downregulating focal adhesion kinase (FAK) and Akt phosphorylation. Mechanistic analyses indicate that the miR-130 family directly targets phosphatase and tensin homolog deleted from chromosome 10 (PTEN), resulting in the upregulation of FAK and Akt phosphorylation. In clinical bladder cancer specimens, downregulation of PTEN was found to be closely correlated with miR-130 family expression levels. Overall, the miR-130 family has a crucial role in malignant progression of bladder cancer and thus the miR-130 family could be a promising therapeutic target for invasive bladder cancer.

Regulator of cullins-1 expression knockdown suppresses the malignant progression of muscle-invasive transitional cell carcinoma by regulating mTOR/DEPTOR pathway.

Background:Regulator of cullins-1 (ROC1) is a key subunit in the cullin–RING ligase (CRL) protein complex. Our previous study indicated that ROC1 was essential for bladder cancer cell survival and that ROC1 knockdown inhibited CRL activity, triggering G2 phase arrest and senescence. However, the role of ROC1 in the malignant progression of bladder cancer remained unknown.Methods:ROC1 expression in cancer cells was knocked down by siRNA silencing. The effects of ROC1 silencing were evaluated by in vitro assays for cell migration and by an in vivo mouse metastasis model. Epithelial–mesenchymal transition (EMT) induction was evaluated by immunofluorescence staining and western blotting of EMT-associated proteins. ROC1 expression in human tumours was further evaluated by immunohistochemical analysis.Results:ROC1 knockdown suppresses bladder cancer cell migration by inhibiting EMT. ROC1 knockdown inhibited EMT by inhibiting mammalian target of rapamycin (mTOR) activity via the accumulation of the mTOR-inhibitory protein DEPTOR, a CRL substrate. DEPTOR knockdown partially rescued ROC1 knockdown-inhibited EMT and the ROC1-induced inhibition of cancer cell migration. Furthermore, in vivo studies using a nude mouse metastasis model confirmed the in vitro data. Finally, tissue microarray analysis of clinical bladder cancer specimens indicated a positive correlation between ROC1 expression and EMT.Conclusions:ROC1 has an important role in the malignant progression of bladder cancer via the mTOR/DEPTOR pathway. ROC1 may serve as a novel therapeutic target for the treatment of muscle-invasive transitional cell carcinoma.

Development and Characterization of Bladder Cancer Patient-Derived Xenografts for Molecularly Guided Targeted Therapy

BackgroundThe overarching goal of this project is to establish a patient-derived bladder cancer xenograft (PDX) platform, annotated with deep sequencing and patient clinical information, to accelerate the development of new treatment options for bladder cancer patients. Herein, we describe the creation, initial characterization and use of the platform for this purpose.Methods and FindingsTwenty-two PDXs with annotated clinical information were established from uncultured unselected clinical bladder cancer specimens in immunodeficient NSG mice. The morphological fidelity was maintained in PDXs. Whole exome sequencing revealed that PDXs and parental patient cancers shared 92–97% of genetic aberrations, including multiple druggable targets. For drug repurposing, an EGFR/HER2 dual inhibitor lapatinib was effective in PDX BL0440 (progression-free survival or PFS of 25.4 days versus 18.4 days in the control, p = 0.007), but not in PDX BL0269 (12 days versus 13 days in the control, p = 0.16) although both expressed HER2. To screen for the most effective MTT, we evaluated three drugs (lapatinib, ponatinib, and BEZ235) matched with aberrations in PDX BL0269; but only a PIK3CA inhibitor BEZ235 was effective (p<0.0001). To study the mechanisms of secondary resistance, a fibroblast growth factor receptor 3 inhibitor BGJ398 prolonged PFS of PDX BL0293 from 9.5 days of the control to 18.5 days (p<0.0001), and serial biopsies revealed that the MAPK/ERK and PIK3CA-AKT pathways were activated upon resistance. Inhibition of these pathways significantly prolonged PFS from 12 day of the control to 22 days (p = 0.001). To screen for effective chemotherapeutic drugs, four of the first six PDXs were sensitive to the cisplatin/gemcitabine combination, and chemoresistance to one drug could be overcome by the other drug.ConclusionThe PDX models described here show good correlation with the patient at the genomic level and known patient response to treatment. This supports further evaluation of the PDXs for their ability to accurately predict a patient’s response to new targeted and combination strategies for bladder cancer.